KLF12 Regulates Mouse NK Cell Proliferation

Abstract

NK cells are innate lymphocytes that play an integral role in tumor rejection and viral clearance. Unlike their other lymphocyte counterparts, NK cells have the unique ability to recognize and lyse target cells without prior exposure. However, there are no known NK cell-specific genes that are exclusively expressed by all NK cells. Therefore, identification of NK cell-specific genes would allow a better understanding of why NK cells are unique cytotoxic lymphocytes. From the Immunological Genome (ImmGen) Consortium studies, we identified kruppel-like factor 12 (Klf12), encoding a novel transcription factor, preferentially expressed in C57BL/6 mouse NK cells. KLF12 was dispensable for NK cell development, IFN-γ production, degranulation, and proliferation in Klf12 knockout mice. RNA-sequencing analysis revealed increased expression of Btg3, an antiproliferative gene, in KLF12-deficient NK cells compared with wild-type NK cells. Interestingly, competitive mixed bone marrow chimeric mice exhibited reduced development of KLF12-deficient NK cells, altered IFN-γ production and degranulation, and impairment of NK cell proliferation in vitro and in vivo in response to mouse CMV infection. KLF12-deficient NK cells from bone marrow chimeric mice also expressed higher levels of the IL-21R, which resulted in increased IL-21R signaling and correlated with greater inhibition of NK cell proliferation. Furthermore, IL-21 induced Btg3 expression, which correlated with arrested NK cell maturation and proliferation. In summary, we found that KLF12 regulates mouse NK cell proliferation potentially by regulating expression of Btg3 via IL-21.

FIGURE 1.

KLF12 is preferentially expressed in mature mouse NK cells. Quantitative PCR of Klf12 expression in (A) sorted splenic populations defined as CD4⁺ T cells: TCRβ⁺NK1.1⁻CD8⁻CD4⁺CD25⁻; CD8⁺ T cells: TCRβ⁺NK1.1⁻CD4⁻CD8⁺CD25⁻; CD19⁺ B cells: TCRβ⁻NK1.1⁻CD19⁺; NK cells: TCRβ⁻NK1.1⁺, and (B) sorted NK cell developmental stages in bone marrow defined as NKP: CD3⁻CD8⁻CD19⁻Ter119⁻Gr1⁻NK1.1⁻DX5⁻CD122⁺, iNK: CD122⁺NK1.1⁺DX5⁻, mNK: CD122⁺NK1.1⁺DX5⁺. Developmental stages in spleen defined as iNK: TCRβ⁻NK1.1⁺CD27⁺CD11b⁻, DP: TCRβ⁻NK1.1⁺CD27⁺CD11b⁺, mNK: TCRβ⁻NK1.1⁺CD27⁻CD11b⁺. Data are representative of 2 experiments (n = 3 mice/experiment).

FIGURE 2.

Targeted disruption of the Klf12 locus. (A) Schematic of the targeting strategy into the Klf12 locus to generate Klf12 conditional knockout mice after excision of the lacZ and neomycin cassettes by Flippase recombination. (B) Southern blot of gDNA from selected ES cell clones digested with EcoRV and hybridized to a probe indicated in (A). The 7.68 kb band is the WT allele and the 6.44 kb band is the targeted allele. (C) Long range PCR of genomic (g)DNA from heterozygous (lane 1) and WT (lane 2) mice amplifying the 3’ arm of the targeting vector with primers indicated in (A). The 5.7 kb band is the targeted allele. (D) PCR of mouse gDNA from indicated genotypes. (E) Quantitative PCR amplifying exons 2–3 (left) or exons 5–6 (right) of Klf12. (F) PCR sequence of Klf12 cDNA from WT or Klf12^F/F β-actin Cre⁺ mice. The premature stop codon is highlighted in grey. (E-F) Data are representative of n = 3 mice/genotype.

FIGURE 3.

NK cell development and function are normal in KLF12-deficient mice. (A) Percentage and total cell numbers of bone marrow (left panels) and splenic (middle panels) NK cell developmental subsets in Klf12^+/+ (circle), Klf12^F/+ (square), and Klf12^F/F (triangle) mice. DN NK cell developmental subset defined as TCRβ⁻NK1.1⁺CD27⁻CD11b⁻. NK receptor expression (right panels) gated on splenic TCRβ⁻NK1.1⁺ NK cells. (B) IFN-γ and CD107a staining gated on TCRβ⁻DX5⁺ NK cells after in vitro stimulation for 6 hr. (A-B) Data are representative of 3 experiments (n = 3 mice/genotype/experiment). (C) Proliferation of TCRβ⁻NK1.1⁺ and TCRβ⁻NK1.1⁺Ly49H⁺ NK cells after in vitro stimulation. Data are representative of 2 experiments (n = 3 mice/genotype/experiment). (D) Percentage of Ly49H⁺ cells gated on TCRβ⁻NK1.1⁺ NK cells in the blood and viral titers in the blood and oral lavage following MCMV infection. Data are representative of 2 experiments (n = 2–4 mice/genotype/experiment).

FIGURE 4.

KLF12-deficient NK cells intrinsically express more Btg3 transcripts. (A) Gene set enrichment analysis of Klf12^+/+ and Klf12^F/F splenic NK cells defined as CD3⁻NK1.1⁺ (n = 3 mice/genotype). Quantitative PCR of Btg3 expression in (B) sorted splenic populations defined as CD4⁺ T cells: TCRβ⁺NK1.1⁻CD8⁻CD4⁺CD25⁻; CD8+ T cells: TCRβ⁺NK1.1⁻CD4⁻CD8⁺CD25⁻; CD19⁺ B cells: CD3⁻NK1.1⁻CD19⁺; NK cells: CD3⁻NK1.1⁺, and (C) sorted splenic TCRβ⁻NK1.1⁺ NK cells from Klf12^+/+ and Klf12^F/F BM chimeric mice. (B-C) Data are representative of 2 experiments (n = 2–3 mice/genotype/experiment for (B)) and (n = 3 mice/experiment for (C)). Full transcription datasets are available at gene expression omnibus GSE128962.

FIGURE 5.

KLF12-deficient NK cells are competitively disadvantaged. (A) Total cell numbers of bone marrow (left panel) and splenic (middle panel) NK cell subsets in bone marrow chimeras at a 1:1 ratio of Klf12^+/+ (circle) and Klf12^F/F (triangle) cells. NK receptor expression (right panel) gated on splenic TCRβ⁻NK1.1⁺ NK cells. Data are representative of 6 experiments (n = 3 mice/experiment). (B) IFN-γ and CD107a staining gated on TCRβ⁻DX5⁺ NK cells after in vitro stimulation for 6 hr. Data are representative of 3 experiments (n = 3 mice/experiment). (C) Proliferation of TCRβ⁻NK1.1⁺ and TCRβ⁻NK1.1⁺Ly49H⁺ NK cells after in vitro stimulation. Data are representative of 8 experiments (n = 2–3 mice/experiment). (D-E) Number (top panels) and relative change in number (bottom panels) of TCRβ⁻NK1.1⁺Ly49H⁺ NK cells in the blood during MCMV infection in (D) bone marrow chimeras and (E) adoptive transfer model where a 1:1 ratio of Klf12^+/+ and Klf12^F/F TCRβ⁻NK1.1⁺Ly49H⁺ NK cells were transferred into Ly49H-deficient hosts one day prior to MCMV infection. Data are representative of (D) 3 experiments (n = 3–6 mice/experiment) or (E) 6 experiments (n = 3–5 mice/experiment). *p < 0.05, ** p < 0.005.

FIGURE 6.

KLF12-deficient NK cells from BM chimeric mice have decreased CD132 expression but normal responsiveness to IL-15. (A) Representative histogram of CD132 and CD122 expression on Klf12^+/+ (black line) and Klf12^F/F (grey line) splenic TCRβ⁻NK1.1⁺ NK cells. MFI of CD132 and CD122 expression on splenic TCRβ⁻NK1.1⁺ NK cells (left panel) and NK cell developmental subsets (right panel). Data are representative of 2 experiments (n = 3 mice/experiment). (B) Percentage and MFI of pSTAT5 in splenic TCRβ⁻NK1.1⁺ NK cells upon ex vivo IL-15 stimulation. Data are representative of 5 experiments (n = 2–3 mice/experiment). *p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001.

FIGURE 7.

IL-21R expression and signaling are increased in KLF12-deficient NK cells from mixed BM chimeric mice. (A) Representative histogram of IL-21R expression on Klf12^+/+ (black line) and Klf12^F/F (grey line) splenic TCRβ⁻NK1.1⁺ NK cells. The grey-filled histogram is the fluorescence minus one control. MFI of IL-21R expression on splenic TCRβ⁻NK1.1⁺ NK cells (left panel) and NK cell developmental subsets (right panel). Data are representative of 2 experiments (n = 2 mice/experiment). (B) Percentage and MFI of pSTAT3 in splenic TCRβ⁻NK1.1⁺ NK cells (left panels) upon IL-21 stimulation ex vivo and NK cell developmental subsets (right panels) upon 0.125 ng/ml IL-21 stimulation ex vivo for 15 min. Data are representative of 2 experiments (n = 2 mice/experiment). (C) Percentage of splenic TCRβ⁻NK1.1⁺ NK cells (left panels) and NK cell developmental subsets (right panels) after 7 days of in vitro culture in 10 ng/ml IL-15 in the absence or presence of 100 ng/ml IL-21. Data are representative of 2 experiments (n = 2 mice/experiment). *p < 0.05, ** p < 0.005.

FIGURE 8.

IL-21 induced Btg3 expression in NK cells. Quantitative PCR of Btg3 expression in enriched C57BL/6 wild-type splenic TCRβ⁻NK1.1⁺ NK cells cultured with 100 ng/ml of IL-21 for the indicated times. Expression of Btg3 was normalized to 0 hr. Data are representative of 3 experiments (n = 2–3 mice/experiment). *p < 0.05, ** p < 0.005.